P1B-type ATPases transport soft metal ions across membranes. These pumps maintain homeostasis of essential trace metals like copper, zinc, cobalt and nickel, and mediate resistance to toxic metals such as lead, cadmium and silver. Soft metals have highly distinct chemistry, which gives rise to unique challenges faced by soft metal transporters. These include binding and release of soft metal ions, which tend to bind extremely tightly to their ligands, and to distinguish between different soft metal ions like the divalent zinc, copper and cobalt ions, which have similar sizes and properties. For example, in humans, zinc transporters have to distinguish between the highly similar zinc and cupric ions. The focus of this study, ZntA, is a P1B-type ATPase from Escherichia coli that confers resistance to toxic levels of zinc, cadmium and lead. It is an excellent model system to study P1B-type ATPases, both in vivo due to the simple bacterial genetics involved, and in vitro since it has been purified, biochemically characterized and tools have been developed to study metal affinity, metal binding kinetics and coordination structure. The major goals of this application are to understand the transport mechanism of P1 B-type ATPases in relation to their structure and to elucidate the molecular basis of metal specificity in soft-metal transporters in general. Specific aims include kinetic studies of metal binding and release steps, the mechanism by which the N-terminal metal binding site increases activity, the role of conserved polar and non-polar residues in the membrane domain in defining metal selectivity and to obtain structural information using EXAFS, fluorescence and X-ray techniques. The results from these studies will have an impact on understanding the binding and transport of the highly toxic metal, lead, in biological systems. Valuable information will be obtained regarding the selectivity and transport of zinc, an extremely important metal ion in biology that has roles as structural and catalytic components of proteins. Zinc also acts as a neuromodulator and stabilizes hormones. Defects in zinc transporters in humans have been implicated in diseases such as prostate cancer. ZntA is also a good biochemical model system for Wilson's and Menkes'disease associated copper transporters in humans, defects in which lead to lethal neurodegenerative diseases, vascular complications and connective tissue defects.